Lipase Preparations Research Articles

Immobilization of Lipase from Thermomyces Lanuginosus and Its Glycerolysis Ability in Diacylglycerol Preparation.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose(DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80°C. Therefore, D101 was chosen as the supporting material of the target protein. The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Extending the computational and experimental analysis of lipase active site selectivity.

Molecular docking is an important computational analysis widely used to predict the interaction of enzymes with several starting materials for developing new valuable products from several starting materials, including oils and fats. In the present study, molecular docking was used as an efficient in silico screening tool to select biocatalysts with the highest catalytic performance in butyl esters production in a solvent-free system, an eco-friendly approach, via direct esterification of free fatty acids from Licuri oil with butanol. For such purpose, three commercial lipase preparations were used to perform molecular docking studies such as Burkholderia cepacia (BCL), Porcine pancreatic (PPL), and Candida rugosa (CRL). Concurrently, the results obtained in BCL and CRL are the most efficient in the esterification process due to their higher preference for catalyzing the esterification of lauric acid, the main fatty acid found in the licuri oil composition. Meanwhile, PPL was the least efficient because it preferentially interacts with minor fatty acids. Molecular docking with the experimental results indicated the better performance in the synthesis of esters was BCL. In conclusion, experimental results analysis shows higher enzymatic productivity in esterification reactions of 1294.83μmol/h.mg, while the CRL and PPL demonstrated the lowest performance (189.87μmol / h.mg and 23.96μmol / h.mg, respectively). Thus, molecular docking and experimental results indicate that BCL is a more efficient lipase to produce fatty acids and esters from licuri oil with a high content of lauric acid. In addition, this study also demonstrates the application of molecular docking as an important tool for lipase screening to achieve more sustainable production of butyl esters with a view synthesis of biolubricants.

Bioimprinted lipase-catalyzed synthesis of medium- and long-chain structured lipids rich in docosahexaenoic acid for infant formula.

Medium- and long-chain structured lipids (MLSLs) rich in docosahexaenoic acid (DHA) were obtained in shorter reaction time by acidolysis of single-cell oil (DHASCO) from Schizochytrium sp. with caprylic acid (CA) using a lipase bioimprinted with fatty acids as a catalyst. The conditions for preparation of the bioimprinted lipase for the acidolysis reaction were firstly optimized and the activity of the obtained lipase was 2.17 times higher than that of the non-bioimprinted. The bioimprinted lipase was then used as a catalyst and the reaction conditions were optimized. Under the optimal conditions, the equilibrium could be achieved in 4h, and the total and sn-1,3 CA contents in the product were 29.18% and 42.34%, respectively, and the total and sn-2 DHA contents were 46.26% and 70.12%, respectively. Such MLSLs rich in sn-1,3 CA and sn-2 DHA are beneficial for DHA absorption, and thus have potential for use in infant formula.

Lipase as a Chiral Selector Immobilised on Carboxylated Single-Walled Carbon Nanotubes and Encapsulated in the Organic Polymer Monolithic Capillary for Nano-High Performance Liquid Chromatography Enantioseparation of Racemic Pharmaceuticals.

Herein, we report the preparation of lipase immobilised on single-walled carbon nanotubes (SWCNTs) as an enantioselector for capillary monolithic columns and their application in the chiral separation of racemic pharmaceuticals. The columns were prepared through the encapsulation of functionalised SWCNTs (c-SWCNTs) within an organic monolithic polymer, followed by the immobilisation of lipase over the obtained monolith, over a three-day (L1) and five-day (L2) period. The prepared columns were tested for the enantioselective nano-HPLC separation of 50 racemic drugs. A suitable resolution was achieved for 25 drugs using nano-RP-HPLC conditions for both the L1 and L2 capillaries, while no specific resolution was detected under normal-phase HPLC conditions. The developed c-SWCNT-lipase-based polymeric monolithic capillaries are a promising expansion for separating pharmaceutical enantiomers' using nano-HPLC.

Chemo-Enzymatic Synthesis of Enantiopure β-Blocker (S)-Metoprolol and Derivatives

(S)-Metoprolol, ((2 S)-1-[4-(2-methoxyethyl)phenoxy]-3-(propan-2-ylamino)propan-2-ol has been synthesised in 99% ee with high yield by a four step chemoenzymatic protocol. Several preparations of Candida antarctica lipase B have been screened in a kinetic resolution of the secondary chlorohydrin 1-chloro-3-(4-(2-methoxyethyl)phenoxy)propan-2-ol. We here report specific rotation values of the enantiopure chlorohydrins from the enzyme catalysed kinetic resolution, in addition to a specific rotation value for (S)-metoprolol, which determines the absolute configuration of the drug, and also the absolute configuration of the chlorohydrin enantiomers.

Preparation and Catalytic Properties of Lipase Immobilized on Epoxy‐functionalized Starch Nanoparticles

AbstractAs a natural macromolecular material, starch is an ideal carrier for enzyme immobilization because of its widely available source, easy regeneration and excellent biodegradability. However, the natural starch cannot be directly used for immobilization due to the large particle size and excessive hydrophilicity. In this paper, the epoxy groups were grafted onto esterified starch by bifunctional reagents. The grafting efficiency of epoxy groups was increased by starvation drop addition. The changes of contact angle and particle size are conducive to the adsorption of immobilized enzyme at the water oil interface in the subsequent enzyme catalytic reaction, hence improve of the lipase activity. The maximum amount of lipase mobilized on such modified starch was ∼143.7 mg/g, which was near 40 mg/g higher than that of those from direct crosslinking. The immobilized enzymes showed excellent resistance to organic solvents and good reusability. Immobilization of lipases on epoxy‐functionalized starch nanoparticles can potentially improves the possibility of enzymes industrial application.

Lipase‐mediated plant oil hydrolysis—Toward a quantitative glycerol recovery for the synthesis of pure allyl alcohol and acrylonitrile

AbstractPlant based triacylglycerides are a sustainable feedstock for the chemical, bioenergy, food and cosmetic sector. While fatty acids conversion has been intensively studied, processes for glycerol valorization have been scarce. In contrast to chemical hydrolysis of plant triacylglycerides enzymatic hydrolysis methods provide a cleaner glycerol stream. This study focuses on the selection of a commercial lipase capable of quantitative hydrolysis of rapeseed‐ and high oleic sunflower oils. Enzymatic process using only water as the reaction medium allows simplified recovery of pure glycerol. Among the six commercial lipase preparations tested, Candida rugosa lipase was identified as the most effective biocatalyst. Catalytic behavior in buffer and pure water was equivalent. Glycerol generated using a washed lipase was recovered by just three purification steps. The FTIR spectrum of the recovered glycerol was equivalent to pure glycerol standard. Over the entire recovery process, 82%–88% of the theoretical glycerol yield could be obtained. Purified glycerol was further didehydroxylated to allyl alcohol by a formic acid mediated open distillation process. The enriched allyl alcohol had a quality, which allows to use it for the synthesis of bio‐based acrylonitrile.

Thermally Stable and Reusable Silica and Nano-Fructosome Encapsulated CalB Enzyme Particles for Rapid Enzymatic Hydrolysis and Acylation.

This study reports the preparation of silica-coated and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and a demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3-100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. The catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated using the Michaelis-Menten equation and Lineweaver-Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and a temperature of 35 °C. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated via an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.

Comparative study of lipase preparations for enzymatic degumming of sunflower oil

Comparative study of lipase preparations for enzymatic degumming of sunflower oil

Exogenous Penicillium camemberti Lipase Preparation Exerts Prebiotic-like Effects by Increasing Cecal Bifidobacterium and Lactobacillus Abundance in Rats

Penicillium camemberti is used in cheese production; however, its health benefits remain to be elucidated. We previously found that supplemental Aspergillus-derived lipase preparation exerts a strong bifidogenic effect in rats fed a high-fat diet. This study investigated the effects of the feeding of a diet containing a 0.2% or 0.4% Penicillium camemberti-derived lipase preparation (PCL) for two weeks on the cecal microbiota in rats. According to 16S rRNA gene sequencing analysis, both PCL supplements significantly (p < 0.05) affected the cecal microbial community. At the genus level, supplemental 0.4% PCL significantly increased the relative abundance of beneficial bacteria such as Bifidobacterium, Lactobacillus, and Collinsella (127-fold, 6-fold, and 193-fold increase, respectively). The abundance of these bacteria in the 0.2% PCL group was between that of the control and 0.4% PCL groups. Notably, the effects of supplemental 0.4% PCL on modulating the abundance of these bacteria matched the effects observed in studies on typical prebiotic oligosaccharides. PICRUSt analysis revealed that PCL supplements significantly modulated the relative abundance of bacterial genes associated with 27 metabolic pathways, some of which were similar to those reported for prebiotic oligosaccharides. This study provides the first evidence indicating that supplemental PCL exerts prebiotic-like effects by modulating the abundance of the gut microbiota.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

Expanding Access to Optically Active Non-Steroidal Anti-Inflammatory Drugs via Lipase-Catalyzed KR of Racemic Acids Using Trialkyl Orthoesters as Irreversible Alkoxy Group Donors

Studies into the enzymatic kinetic resolution (EKR) of 2-arylpropanoic acids (‘profens’), as the active pharmaceutical ingredients (APIs) of blockbuster non-steroidal anti-inflammatory drugs (NSAIDs), by using various trialkyl orthoesters as irreversible alkoxy group donors in organic media, were performed. The enzymatic reactions of target substrates were optimized using several different immobilized preparations of lipase type B from the yeast Candida antarctica (CAL-B). The influence of crucial parameters, including the type of enzyme and alkoxy agent, as well as the nature of the organic co-solvent and time of the process on the conversion and enantioselectivity of the enzymatic kinetic resolution, is described. The optimal EKR procedure for the racemic profens consisted of a Novozym 435-STREM lipase preparation suspended in a mixture of 3 equiv of trimethyl or triethyl orthoacetate as alkoxy donor and toluene or n-hexane as co-solvent, depending on the employed racemic NSAIDs. The reported biocatalytic system provided optically active products with moderate-to-good enantioselectivity upon esterification lasting for 7–48 h, with most promising results in terms of enantiomeric purity of the pharmacologically active enantiomers of title APIs obtained on the analytical scale for: (S)-flurbiprofen (97% ee), (S)-ibuprofen (91% ee), (S)-ketoprofen (69% ee), and (S)-naproxen (63% ee), respectively. In turn, the employment of optimal conditions on a preparative-scale enabled us to obtain the (S)-enantiomers of: flurbiprofen in 28% yield and 97% ee, ibuprofen in 45% yield and 56% ee, (S)-ketoprofen in 23% yield and 69% ee, and naproxen in 42% yield and 57% ee, respectively. The devised method turned out to be inefficient toward racemic etodolac regardless of the lipase and alkoxy group donor used, proving that it is unsuitable for carboxylic acids possessing tertiary chiral centers.

Application of undecanedicarboxylic acid to prepare cross-linked enzymes (CLEs) of Rhizomucor miehei lipase (RML); Selective enrichment of polyunsaturated fatty acids

We have introduced 1,11-undecanedicarboxylic acid as a new cross-linker to prepare cross-linked preparation of Rhizomucor miehei lipase (RML) from its soluble form by applying an isocyanide-based multi-component reaction. This one-step cross-linking of soluble enzyme allowed skipping the prior step of enzyme aggregation or crystallization which is inevitable in the traditional methods of cross-linking. High immobilization yields were achieved up to 92% in an extremely mild condition. The addition of bovine serum albumin (BSA) as a feeder protein affected the final properties of RML by producing 2.5-fold higher specific activity compared to the specific activity of free RML. Furthermore, dynamic light scattering analysis (DLS) showed that by applying BSA, the cross-linked enzymes (CLEs) particle sizes of 1.75–3.91 µm were obtained. Considerable improvement in the thermal and co-solvent stability was observed, in particular for the derivative obtained by immobilization in the presence of BSA. This derivative remained considerably active at 60 °C by keeping 79% of its initial activity after 2 h of incubation and retained 75% and 86% of its activity after 24 h incubation in the presence of 20% of methanol and ethanol, respectively. The cross-linked preparations were then applied for the selective hydrolysis of the essential omega-3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from fish oil. The results showed that the immobilized derivatives tend to preferentially hydrolyze EPA.

Aspergillus-Derived Cellulase Preparation Exhibits Prebiotic-like Effects on Gut Microbiota in Rats

Aspergillus-derived cellulase, which is involved in the production of Aspergillus-fermented foods, has been employed in the food and animal feed industries. However, the effect of dietary Aspergillus cellulase on health is poorly understood. Previously, we discovered that supplemental Aspergillus-derived protease and lipase preparations had substantial bifidogenic effects on rats fed a high-fat diet. Therefore, this study reports on the effects of a 0.1% dietary Aspergillus-derived cellulase preparation (CEL) on the gut microbiota of rats fed a high-fat diet. Gene sequencing analysis of 16S rRNA revealed that CEL treatment markedly affected the microbiota profiles of the cecal contents (p < 0.05). Notably, CEL markedly increased the relative abundance (RA) of typical probiotics, such as Bifidobacterium and Lactobacillus, at the genus level (28- and 5-fold, respectively, p < 0.05). Similarly, at the family level, CEL treatment significantly increased the RA of Bifidobacteriaceae and Lactobacillaceae (p < 0.05). Furthermore, CEL increased the RA of other genera, such as Collinsella and Enterococcus, but decreased the RA of Oscillospira, Dorea and Coprobacillus (p < 0.05). The effects on these genera are similar to those reported for typical prebiotic oligosaccharides. Overall, this study demonstrates the prebiotic-like effects of dietary CEL by significantly increasing Bifidobacterium and Lactobacillus abundance.

Supplemental Aspergillus Lipase and Protease Preparations Display Powerful Bifidogenic Effects and Modulate the Gut Microbiota Community of Rats

Aspergillus-derived protease and lipase, which are involved in the production of Aspergillus-fermented foods, are consumed as digestive enzyme supplements. A marked bifidogenic effect of supplemental Aspergillus protease preparation (AP) in rats fed with a high-fat diet was identified. This study was conducted to examine whether the consumption of Aspergillus-derived lipase exerts similar bifidogenic effect. Rats were fed diets supplemented with either an Aspergillus-derived lipase preparation (AL) or AP at 0.1% for two weeks. 16S rRNA gene sequencing analysis indicated that supplemental AL and AP markedly influenced cecal microbial community. At the phylum level, treatment with AL and AP resulted in a lower relative abundance of Firmicutes and Bacteroidetes, but a higher relative abundance of Actinobacteria and Proteobacteria than the control rats (p < 0.05). At the genus level, AL and AP remarkedly elevated the relative abundances of Bifidobacterium, Collinsella, and Enterococcus, but significantly reduced those of Oscillospira, Dorea, and Coprobacillus (p < 0.05). These modulations were similar to those reported by several studies with typical prebiotic oligosaccharides. Notably, the bifidogenic effect of AL was much greater than that of AP. Our results show that the two different Aspergillus-derived preparations, AL and AP, have strong bifidogenic effects and can change the microbiota’s composition.

Eco-friendly production of trimethylolpropane triesters from refined and used soybean cooking oils using an immobilized low-cost lipase (Eversa>® Transform 2.0) as heterogeneous catalyst

Trimethylolpropane triesters – TMPTEs (biolubricants) have been produced through enzymatic hydroesterification of refined soybean (RSO) or used soybean cooking (USCO) oils. In the first step, enzymatic hydrolysis has been performed using a crude free lipase extract from Candida rugosa (CRL) to produce free fatty acids (FFAs). Complete hydrolysis of the oils was achieved after 3 h of reaction. Then, CRL and the lipases from Thermomyces lanuginosus (Lipolase® 100L and Eversa® Transform 2.0) and Pseudomonas fluorescens (Amano AK) were immobilized via interfacial activation on polystyrene-divinylbenzene (PSty-DVB) beads. High enzyme loaded biocatalysts (immobilization yield above 85%) were obtained for all lipase preparations using an initial protein loading of 40 mg g−1. Then, free lipases (liquid or powder forms) or immobilized biocatalysts were employed to produce TMPTEs via enzymatic esterification of the produced FFAs with trimethylolpropane (TMP) in eco-friendly processes (solvent-free systems). The new heterogeneous biocatalyst prepared in this study (immobilized Eversa® Transform 2.0) was the most active biocatalyst in TMPTEs production and maximum OH conversion of ≈97% using both FFA sources was achieved after 4 h of reaction conducted in open reactors at 55 °C, TMP:FFA ratio of 1:3.25, 240 rpm, and 15% of mass of biocatalyst per mass of starting materials. Under similar conditions, OH conversion of ∼25% after 5 h of reaction was obtained using Novozym® 435 as biocatalyst. Immobilized Eversa® Transform 2.0 retained above 90% of its original activity after 13 consecutive reaction cycles. The produced TMPTEs presented good cold temperature properties (pour point values between −11 and −9 °C).

Immobilization and bioimprinting strategies to enhance the performance in organic medium of the metagenomic lipase LipC12

The present work evaluates the immobilization of LipC12 on different supports in tandem with bioimprinting technique, in order to improve its activity and stability in organic medium. Oleic acid was selected as the bioimprinting molecule. The immobilized LipC12 was applied in the synthesis of pentyl oleate by esterification reaction and in the production of fatty acids, mono, and diglycerides via hydrolysis of triacylglycerols, in n-heptane reaction media. For all immobilized lipase preparations, an increase in the conversion of oleic acid to pentyl oleate was observed when immobilization in tandem with bioimprinting treatment was carried out versus immobilization without bioimprinting. The highest conversions were achieved using LipC12 immobilized on hydrophobic supports. The reuse potential of the immobilized preparations was evaluated. The preparations were used in eight successive cycles of esterification reactions and the best results were obtained for LipC12 immobilized on Immobead 150 and chitosan. The activity for the hydrolysis of soybean oil was improved by bioimprinting treatment only for LipC12 immobilized on commercial polypropylene and Accurel MP-1000. LipC12 immobilized on hydrophilic supports or on Immobead150 could be used to hydrolyze tricaprylin to obtain diglycerides with a high proportion of 1,2-diglycerides in reaction times as short as 30 min.

Hydrolysis of vegetable and microbial oils catalyzed by a solid preparation of castor bean lipase

Motivated by the search for inexpensive biocatalysts , this study aimed to investigate the potential of lipase from castor bean seeds for hydrolysis of high-acidity oils. Biochemical properties, kinetics, and thermal stability were investigated in reactions with vegetable (macaw pulp and kernel) and microalgal ( Dunaliella salina , Nannochloropsis gaditana , and Chlorella minutissima ) oils. Enzyme performance was optimal at pH 4.5 and 35 °C. Thermal stability tests revealed a half-life of 9.6 h at 35 °C and apparent V max and K m values of 181.82 U g −1 and 215.15 mmol L −1 , respectively. The rate of hydrolysis was influenced by oil type, fatty acid composition , and oil concentration in the reaction medium. Castor bean lipase showed higher specificity toward vegetable oils rich in polyunsaturated fatty acids . The highest hydrolysis percentage of macaw pulp oil (53.55 ± 2.03%) was obtained at an oil/water weight ratio of 1:4 after 3 h of reaction. Lower performances were achieved with microalgal oils (31–43%), attributed to the presence of pigments , particularly chlorophyll, which has a negative effect on biocatalyst activity. The highest hydrolysis percentage was obtained using D. salina oil (43%) in 4 h of reaction. N. gaditana and C. minutissima oils had a hydrolysis percentage of 34 and 31%, respectively. D. salina oil had a similar fatty acid composition to macaw pulp oil, with 37.5% oleic acid and 15.4% linoleic acid . Nevertheless, the hydrolysis percentage of N. salina oil was 10% lower than that of macaw pulp oil. The low-cost solid preparation of castor bean lipase showed satisfactory performance in catalyzing the hydrolysis of vegetable oils. This economically attractive strategy can be used to obtain free fatty acids from non-edible oils for the production of biodiesel by hydroesterification. • Plant lipases have emerged as a feasible low-cost alternative with high specificity. • Castor bean lipase showed higher specificity toward vegetable and microbial oils. • This attractive strategy can be used in reactions of hydroesterification.